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in Human Macrophages1
Institute of Parasitology, McGill University, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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priming is essential for
macrophage accessory and effector cell functions, we investigated the
effect of IFN- on COX-2 expression in U937 human macrophages
stimulated with IL-1ß. A dose- and time-dependent increase in COX-2
mRNA and protein expression was evoked by IL-1ß, whereas the levels
of COX-1, the constitutively expressed isoform, remained unaltered.
Interestingly, IFN--primed cells showed 40 to 60% lower levels of
COX-2 mRNA, protein expression, and PGE2 production as
compared with nonprimed cells. IFN--priming (50–500 U/ml)
down-regulated COX-2 expression in a time- and dose-dependent fashion.
Furthermore, IFN- inhibited COX-2 gene transcription in
response to IL-1ß but not to LPS. In contrast, the rate of decay of
COX-2 transcripts in nonprimed and primed macrophages was similar
(t1/2 = 3.2 h). The down-regulatory
effect of IFN- on IL-1ß-induced COX-2 expression was abrogated
with cycloheximide. These results highlight a novel mechanism of COX-2
regulation by IFN- at the transcriptional level, which may affect
the outcome of inflammatory and immune
conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2), favoring the
production of Th2-type cytokines (IL-4, IL-5) by human lymphocytes
(3, 4). PGE2 acts in an autocrine manner to
inhibit macrophage accessory and effector cell functions, such as the
expression of MHC molecules (5) or the production of
TNF-
(6).
The rate-limiting step in the synthesis of PGs is the expression
of the enzyme cyclooxygenase (COX).3 This
enzyme catalyzes the conversion of arachidonic acid to
PGH2, the precursor of biologically active PGs. However,
the enzyme is rapidly inactivated after catalytic activity. Therefore,
to achieve a sustained production of PGs, new enzyme must be
synthesized (7). Two isoforms of COX have been described:
COX-1, generally referred to as the "constitutive" isoform and
COX-2, which is readily induced after stimulation with cytokines,
mitogens, and phorbol esters (8, 9, 10). COX-2 is
predominantly expressed in cells involved in inflammatory reactions,
such as macrophages, endothelial cells, and fibroblasts (8, 11, 12). COX isoforms are encoded by two different genes,
thus being independently regulated. Several proinflammatory cytokines
stimulate the expression of COX-2. IL-1 has been shown to induce a
transient transcription of the COX-2 gene in human
endothelial cells (9). IL-1ß enhances COX-2 mRNA
expression in human monocytes (8) and stabilizes COX-2
transcripts in renal mesangial cells (13). TNF- induces
COX-2 expression in osteoblasts and articular chondrocytes (14, 15). Interestingly, TNF- appeared to be a less potent COX-2
inducer than IL-1ß in osteoblasts and mesangial cells (14, 16). Similarly, human monocytes failed to express COX-2 protein
upon stimulation with TNF- (17). We have shown that
only IFN--primed human macrophages express COX-2 mRNA when TNF-
is the triggering signal (18). TGF-ß is able to
moderately induce COX-2 expression in human lung fibroblasts
(12) and it can enhance the expression of COX-2 by phorbol
esters in murine fibroblasts (19). This effect is
apparently cell type dependent, since it was shown that TGF-ß
attenuates the expression of COX-2 induced by endotoxin in murine
macrophages (20). Down-regulatory activities were also
observed with IL-4 and IL-10, which inhibited PG production and COX-2
expression in human monocytes stimulated with LPS as a result of
transcriptional and posttranscriptional regulation
(21).
IFN- produced by T lymphocytes and NK cells plays a major role in
the biology of monocytes/macrophages as the primary
macrophage-activating factor (22). IFN- stimulates
tumor cell cytotoxicity (23), antimicrobial activity
(24), and Ag processing and presentation through increased
expression of MHC molecules (25). However, the effect of
this cytokine on COX-2 expression is still controversial. Studies done
before the description of COX-2 have shown that IFN- could inhibit
PGE2 production by human monocytes and murine peritoneal
macrophages stimulated with IL-1ß (26) or zymosan
(27). On the other hand, it was recently shown that
IFN- has no effect on COX activity and PGE2 production
by human monocytes exposed to LPS (28).
Given the opposite effects that PGE2 and IFN- have on
macrophage functions, we hypothesized that IFN--primed macrophages
would show a differential expression of COX-2 when stimulated by
IL-1ß or LPS. In this study we investigated the effect of
IFN--priming on COX-2 expression mediated by IL-1ß in human
macrophages. Our data indicate that IFN- priming attenuates the
expression of COX-2 mRNA, protein, and PGE2 production
induced by IL-1ß but not by LPS. The inhibitory effect of IFN-
priming on COX-2 mRNA and protein expression occurred in a dose- and
time-dependent manner. IFN- priming down-regulated COX-2gene transcription in response to IL-1ß but not to LPS and had
no effect on the rate of COX-2 mRNA decay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 with L-glutamine was obtained from Life
Technologies (Burlington, Ontario, Canada) and was completed with 100
U/ml of penicillin, 100 µg/ml streptomycin sulfate, 20 mM HEPES
(Sigma, St. Louis MO), and 10% heat-inactivated FCS (HyClone
Laboratories, Logan, UT). The following reagents were purchased from
Sigma: LPS from Escherichia coli, serotype 0111, 4% phenol
extract; PMA, dexamethasone (DEX);
3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT).
Actinomycin D (AD) was obtained from Life Technologies. IL-1ß was
from R&D Systems (Minneapolis, MN) and IFN- was purchased from
Boehringer Mannheim (Laval, Quebec, Canada).
Cells
The human macrophage cell line U937 was maintained and subcultured in complete RPMI 1640. Cells were kept at 37°C in 5% CO2-95% air and harvested at log phase of growth. The cells were adjusted to 1 x 106 cells/ml in the presence of 10 nM PMA, placed in 24-well plates (1 x 106 cells/well), and incubated for 3 days to differentiate into adherent macrophages. The cells were quiesced in fresh complete RPMI 1640 without PMA for 24 h before stimulation with cytokines or LPS. Cell viability was determined by the MTT method (29) and was 90% after 12 h of incubation with AD.
Northern blot
Total RNA was extracted with TRIZOL (Life Technologies)
following the manufacturer’s instructions. Ten micrograms of RNA were
denatured for 1 h at 50°C with glyoxal. Ethidium bromide was
added to each sample before electrophoresis in a 1% agarose gel. The
RNA was transferred to Hybond nylon membranes (Amersham Canada,
Oakville, Ont., Canada) and UV cross-linked. After 3 h of
prehybridization in a buffer containing 50% formamide (Life
Technologies), 0.5% SDS, 5x SSPE (20x SSPE is 3 M NaCl, 0.2 M
NaH2PO4 · H2O, 20 mM EDTA,
pH 7.4), 5x Denhardt’s mixture (50x Denhardt is 1% Ficoll, 1%
polyvinylpyrrolidone, 1% BSA pentax fraction V) and 200 µg/ml of
denatured salmon sperm DNA, the membranes were hybridized at 42°C
overnight with [-32P]dCTP (10 mCi/ml, ICN
Biochemicals, Montreal, Quebec, Canada)-labeled probes by nick
translation (Amersham kit). The membrane was washed once at room
temperature and twice at 55°C with 0.5x SSC (20x SSC is 3 M NaCl
and 0.3 M sodium citrate, pH 7) for 30 min each and exposed for 24
h to an XAR-5 film (Kodak) with an intensifying screen. The probe for
human COX-1 was the 1.8-kb HindIII and
NotI fragment from the 5.8-kb pcDNAhCOX-1 plasmid. The probe
for human COX-2 was the 1.8 kb EcoRI and
ApaI fragment from the pcDNACOX-2 plasmid, a generous gift
from Dr. T. Hla, American Red Cross (11). To ensure
that equal amounts of RNA were analyzed, the blots were stripped (45
min in boiling 0.1% SDS) and probed for actin with the 1.25-kb
PstI fragment of pBA-1 plasmid (30). The blots
were scanned and the densitometric results analyzed with the National
Institutes of Health Image program 1.59 (available from the Internet by
anonymous FTP from zippy.nim.nih.gov or floppy disk from the National
Technical Information service, Springfield, VA; part no.
PB95-500195GEI).
Immunoblot analysis
One million cells were lysed with 100 µl of sample buffer (64
mM Tris-HCl, pH 6.8, 10.25% glycerol, 2% SDS, and 5.12% 2-ME)
containing protease inhibitors (1.5 mM EDTA, 23 µM leupeptin, 14.5
µM pepstatin, 1.53 µM aprotinin, 30 µM
N-p-tosyl-L-lysine chloromethyl ketone
(TLCK), 1 mM PMSF). The whole cell lysates (equivalent to 1 x
105 cells) were separated in a 10% SDS-PAGE and
transferred to a nitrocellulose membrane (Hoeffer System).
Polyclonal Abs prepared against the complete ovine COX-1 and COX-2
proteins (a gift from Dr. G. O’Neill, Merck Frosst, Kirkland, Quebec,
Canada) were used at a 1:5000 dilution. The secondary Ab (donkey
anti-rabbit IgG-horseradish peroxidase) was diluted 1:3000 and
peroxidase activity was visualized by enhanced chemiluminiescence
system (ECL, Amersham Canada) using Kodak XAR-5 film. Purified isoforms
of COX (Cayman Chemicals, Ann Arbor, MI) were used to determine the
specificity of the Abs.
Nuclear run-on assays
Adherent macrophages were washed twice with cold Dulbecco’s
PBS, scraped, and resuspended in Dulbecco’s PBS. Lysis buffer was
added (320 mM sucrose, 5 mM MgCl2, 10 mM Tris-HCl, 1%
Triton X-100, pH 7.5) and the cells (5 x 107) were
kept on ice for 10 min and centrifuged for 15 min at 1300 x
g at 4°C. The nuclear pellets were washed once in lysis
buffer and the nuclei were resuspended in 100 µl of storage buffer
(50 mM Tris-HCl, pH 8.0, 40% glycerol, EDTA 0.1 mM, 5 mM
MgCl2, and 1 mM DTT) and frozen in liquid nitrogen until
used. Elongation of the nascent RNA was done by adding 100 µl of
transcription buffer (20 mM Tris-HCl, pH 8.0, 300 mM KCl, 10 mM
MgCl2, 200 mM sucrose, 48 µM EDTA, 1 mM DTT, 1 mM of each
ribonucleotides of adenosine, guanosine, and cytidine), and 10 µl of
[-32P]uridine triphosphate (10 mCi/ml, ICN
Biochemicals, Montreal, Quebec) to 100 µl of nuclei and incubating at
30°C for 30 min. The reaction was stopped with 1 ml of TRIZOL and the
RNA was extracted and dissolved in 50 µl of water. The amount of
radioactivity in each sample was adjusted to 5 x 106
cpm, and the samples were hybridized onto nitrocellulose membranes
containing 5 µg of pcDNA COX-1, pcDNACOX-2, pBA-1, pcDNA1Amp, and
pBR322 linearized plasmids. The membranes were prehybridized for 3
h at 65°C in hybridization buffer (5x SSC, 5x Denhardt, 0.05% SDS,
0.5 mM EDTA, and 0.1 µg/µl of denatured salmon sperm DNA) and
hybridized for 48 h. Blots were washed three times with 2x SSC
and 0.1% SDS at room temperature for 10 min each time and twice with
0.2x SSC and 0.1% SDS at 60°C for 15 min and exposed for
autoradiography for 15 days. The membranes were scanned and the
densitometric analysis was performed as indicated for Northern
blot.
PGE2 production
PGE2 production was measured by enzyme immunoassay (EIA, Cayman Chemicals) after different times of incubation with the cytokines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on COX-2 mRNA and
protein expression induced by IL-1ß in human macrophages
We have previously shown that TNF- plus IFN- can
moderately enhance COX-2 mRNA levels in PMA-differentiated U937 human
macrophages (18). To determine whether IL-1ß, another
potent proinflammatory cytokine, can modulate COX-2 expression,
PMA-differentiated U937 human macrophages were exposed to increasing
concentrations of IL-1ß during 6 h and the levels of COX-2
transcripts were evaluated afterward. To evaluate the effect of
macrophage priming on COX-2 expression, cells were preincubated for
24 h with 100 U/ml of IFN- and then stimulated with IL-1ß for
6 h. Figure 1
A shows a
dose-dependent increase on the steady state levels of COX-2 mRNA in
both naive and IFN--primed macrophages. The levels of COX-1 mRNA
showed no alteration regardless of the IL-1ß concentration used.
Interestingly, primed macrophages consistently showed lower levels of
COX-2 mRNA expression (40–60%) irrespective of the IL-1ß
concentration used as compared with naive macrophages. LPS (100 ng/ml),
the positive control, stimulated high levels of COX-2 but not COX-1
mRNA expression in both naive and primed macrophages. Dexamethasone
(DEX) completely inhibited COX-2 expression in response to IL-1ß (10
ng/ml) stimulation but had no effect on constitutively-expressed COX-1
mRNA levels. As observed by immunoblot analysis, COX-2 protein was also
inhibited in IFN--primed cells as compared with naive macrophages
(Fig. 1B). However, the levels of COX-2 protein were similar
in both naive and IFN--primed cells in response to LPS stimulation.
These results indicate that IL-1ß is a strong stimulus to enhance
COX-2 mRNA levels in human macrophages. Furthermore, they suggest that
IFN-, at standard concentrations used for macrophage priming, can
inhibit the levels of COX-2 transcripts and the expression of the COX-2
enzyme when IL-1ß is the triggering signal.
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-primed
macrophages
To determine whether the lower levels of COX-2 mRNA observed
above in primed macrophages were due only to a delay in the
transcription, we studied the kinetics of COX-2 mRNA expression in
response to a suboptimal dose of IL-1ß (10 ng/ml). Figure 2A shows that COX-2 mRNA was
detected as early as 2 h after IL-1ß stimulation, reaching
maximum levels after 12 h of incubation in naive macrophages.
Although a similar kinetics of COX-2 mRNA induction was observed in
primed macrophages, the mRNA levels were consistently lower and never
reached the same as in naive macrophages, even after 24 h of
incubation (data not shown). The expression of COX-2 protein was
detected after 6 h in naive macrophages whereas significantly
reduced levels were observed in primed cells throughout the 12-h
incubation period (Fig. 2B). These data indicate a time
dependency for COX-2 mRNA expression, a lag period for the synthesis of
COX-2 protein, and clearly demonstrate the inhibitory effect of IFN-
on COX-2 mRNA and protein expression stimulated by IL-1ß.
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B, PGE2 production was enhanced (2.8-fold)
above control levels only after 12 h of IL-1ß stimulation. In
IFN--primed macrophages, however, 50% lower levels of
PGE2 were produced as compared with naive cells. The amount
of PGE2 released by nonstimulated cells was quantified at
the end of the incubation (12 h) and represents enzyme activity of
COX-1 protein. These results suggest that despite the presence of COX-2
protein at 6 h of stimulation with IL-1ß, PGE2
synthesis is augmented only after 12 h and indicate that IFN-
is able to modulate the levels of PGE2 secreted by
macrophages.
IFN- down-regulates COX-2 mRNA
induction and protein expression in a time- and dose-dependent manner
We next determined if the time of preexposure with IFN- was
critical for inhibiting COX-2 mRNA expression. In this experiment,
macrophages were preincubated with IFN- at different times and then
stimulated with IL-1ß for 6 h. COX-2 mRNA expression was
inhibited up to 47% in macrophages stimulated simultaneously with
IFN- and IL-1ß as compared with IL-1ß-stimulated naive cells
(Fig. 3A). Short preexposure
(2 and 6 h) with IFN- did not further affect COX-2 mRNA levels;
however, following 12 and 24 h preexposure, COX-2 mRNA expression
was significantly decreased (80%). A direct correlation was observed
between COX-2 mRNA levels and protein expression (Fig. 3B).
Based on these results, we then determined whether the concentration of
IFN- was crucial for regulating the expression of COX-2 mRNA and
protein. Accordingly, macrophages were preincubated for 24 h with
increasing concentrations of IFN- and then stimulated with IL-1ß
for 6 h. As shown in Figure 4, A and
B, there was a dose-dependent
effect of IFN- on the expression of COX-2 mRNA and protein
stimulated by IL-1ß. With 500 U/ml of IFN-, COX-2 mRNA levels were
inhibited by 97% (Fig. 4A). These data suggest that IFN-
can modulate the expression of COX-2 mRNA and protein levels in a time-
and dose-dependent manner.
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inhibits the rate of COX-2 gene
transcription mediated by IL-1ß
The steady state levels of COX-2 mRNA in cells are the result of a
balance between the rate of gene transcription and the rate of mRNA
degradation. To determine whether IL-1ß and IFN- were able to
modulate the rate of COX-2 gene transcription, nuclear
run-on analysis was performed as described in Materials and
Methods. Naive and primed macrophages were stimulated for 1 h
in the presence of IL-1ß or LPS. Figure 5 shows that nonstimulated cells
exhibited a basal level of COX-2 gene transcription, which was
significantly enhanced by IL-1ß (2.5-fold) and LPS (4.7-fold).
However, in IFN--primed macrophages stimulated with IL-1ß, the
relative rate of COX-2 gene transcription was inhibited by
37%. IFN- priming did not affect the rate of COX-2 gene
transcription in response to LPS stimulation and is consistent with the
data presented in Figure 1A. These results indicate that the
inhibitory effect of IFN- on COX-2 expression occurs at the
transcriptional level.
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does not alter the rate of COX-2
mRNA degradation
Previous studies (13) have demonstrated that IL-1ß can
stabilize COX-2 mRNA in renal mesangial cells, showing that
posttranscriptional mechanisms modulate COX-2 expression. A similar
effect was observed with IL-1 in human endothelial cells
(9). In contrast, IL-4 and IL-10 inhibit COX-2 expression
by down-regulating the rate of gene transcription, but also by
increasing the rate of COX-2 mRNA degradation (21). To
investigate whether IFN- was also inhibiting the steady state levels
of COX-2 mRNA by destabilizing the transcripts and therefore increasing
their rate of decay, the following experiment was performed. COX-2 mRNA
expression was maximally induced by IL-1ß for 12 h,
transcription was inhibited with AD, and the fate of the COX-2 mRNA
determined after various times. Figure 6A shows that the levels of
COX-2 mRNA remained consistently high in both naive and IFN--primed
macrophages even after 12 h of inhibiting transcription. We
confirmed that AD was inhibiting transcription as naive and primed
cells simultaneously incubated with IL-1ß and AD for 12 h
demonstrated no expression of COX-2 mRNA (Fig. 6A). In the
absence of AD, the levels of COX-2 mRNA also remained elevated even
after 24 h of stimulation with IL-1ß, and COX-2 transcripts in
primed macrophages never reached the same levels as in naive cells
(Fig. 6B). Similar experiments performed in the absence of
IL-1ß during the chase period with and without AD indicated that
active transcription, together with the removal of IL-1ß, was
necessary for the rapid degradation of COX-2 mRNA (data not shown).
Accordingly, to efficiently compare the rate of degradation between the
COX-2 transcripts in naive and primed macrophages, the
t1/2 of the mRNA was measured in the presence of
DEX, which has been shown to promote the degradation of COX-2 mRNA
(31). The results presented in Figure 7 indicate that the half lives of COX-2
transcripts in naive and primed macrophages were similar (3.2 h),
suggesting that IFN- did not affect the expression of COX-2 at the
posttranscriptional level.
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To elucidate whether de novo protein synthesis was required
for the inhibitory effect of IFN- on COX-2 expression, naive and
primed macrophages were incubated with IL-1ß in the presence or
absence of cycloheximide (CHX) for 12 h. Results in Figure 8 show that CHX enhanced COX-2 mRNA
expression equally in naive and primed cells. Moreover, superinduction
of COX-2 mRNA expression was observed in both naive and primed cells
simultaneously incubated with CHX and IL-1ß for 12 h. The
abrogation of the down-regulatory effect of IFN- on COX-2 expression
by CHX suggests the requirement of newly synthesized proteins
(repressors) for such effect. In addition, newly synthesized factors
may also be involved in the degradation of COX-2 mRNA and, in
consequence, CHX can promote COX-2 accumulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(9), TGF-ß (19),
IL-4, and IL-10 (21), have been described as modulators of
COX-2. However, the combined effect of the network of cytokines in the
milieu of inflamed sites on COX-2 is not completely understood. The
objective of this study was to determine the effect of IFN-, a
prominent modulator of macrophage functions, on the expression of COX-2
elicited by IL-1ß, a proinflammatory cytokine. We demonstrated that
IFN--primed macrophages expressed lower levels of COX-2 mRNA
and COX-2 protein, and produced lower amounts of
PGE2. IFN- exerted its inhibitory effect by decreasing
the rate of COX-2 gene transcription and had no effect on
the turnover rate of COX-2 mRNA. The inhibitory effect of IFN- was
specific for IL-1ß but not LPS-induced COX-2 mRNA expression.
IL-1ß strongly enhanced the expression of COX-2 mRNA in a dose-
and time-dependent manner in both naive and IFN--primed macrophages,
but had no effect on the expression of COX-1. A similar pattern of
COX-2 induction was described in naive human monocytes and synoviocytes
(8, 33). We detected COX-2 mRNA as early as 2 h after
stimulation with IL-1ß whereas COX-2 protein was observed after
6 h of incubation. Furthermore, the amount of PGE2
released only increased above control levels after 12 h of
incubation. This delay in PGE2 production has been observed
in LPS-stimulated macrophages (34) and is probably due to
the unavailability of arachidonic acid, the substrate for COX enzymes.
The activation of cytosolic phospholipase A2 by
platelet-derived growth factor has been shown to be crucial for
the synthesis of PGE2 in murine osteoblasts stimulated with
IL-1 (35).
Our results clearly show a down-regulatory effect of IFN- on
COX-2 mRNA and protein expression and PGE2 production by
macrophages. Previous studies have shown a blocking effect of IFN-
on PGE2 production by human monocytes when stimulated with
either LPS or IL-1ß (26). However, in the present study
and those by Endo et al. (28), IFN- did not inhibit
COX-2 when stimulated with LPS. In contrast, IFN- synergizes with
LPS to enhance the expression of COX-2 and the release of
PGE2 in murine macrophages (36). We have also
reported a synergistic effect of IFN- with TNF- for the
expression of COX-2 mRNA in human macrophages (18). These
findings indicate that the cell source and the triggering signal
determine the final outcome of COX-2 expression and PGE2
production.
IFN- down-regulated the expression of COX-2 mRNA and protein
in a dose- and time-dependent manner. Maximal inhibition was observed
after 24 h of preincubation with this cytokine although
simultaneous incubation with IL-1ß also caused a significant decrease
in COX-2 mRNA levels (47%). We then explored the possible role
of IFN- on regulating COX-2 expression at both transcriptional and
posttranscriptional levels. The steady state levels of COX-2
transcripts are the result of a balance between the rate of gene
transcription and the rate of degradation of the mRNA produced. Studies
in renal mesangial cells suggest that cytosolic factors activated after
IL-1ß stimulation can bind to the 3'-untranslated region of the COX-2
transcripts and prolong their t1/2
(13). In our case, a possible mechanism for the
down-regulatory effect of IFN- was that this cytokine could be
promoting a faster degradation of the IL-1ß-induced COX-2 mRNA and,
therefore, reducing the levels detected by Northern blot, analagous to
what has been reported for c-fos mRNA (37).
Experiments with AD showed that transcription inhibition did not
promote the rapid decay of COX-2 transcripts in either naive or primed
macrophages. A rapid decay of COX-2 transcripts occurred only when
IL-1ß was removed after 12 h of stimulation (our unpublished
observations). These observations, and the results with DEX, suggest
that active transcription of certain genes or the removal of the
stimulus (IL-1ß) was necessary for the rapid decay of COX-2 mRNA. The
ability of CHX to promote the accumulation of transcripts also
indicates that COX-2 mRNA expression is strongly regulated at a
posttranscriptional level. This protein synthesis inhibitor may
be preventing the production of factors involved in the rapid
degradation of COX-2 mRNA as it was suggested for endothelial cells
(9).
Ristimakii et al. (31) have recently reported that DEX, a
synthetic glucocorticoid known to inhibit the expression of COX-2 mRNA,
acts by destabilizing the COX-2 transcripts. DEX does not affect the
expression of COX-1 or actin, therefore we used it to specifically
promote the decay of COX-2 mRNA in naive and primed macrophages to
compare their half-lives. With this strategy, we demonstrated a similar
t1/2 of COX-2 transcripts in naive and primed
macrophages, which ruled out a posttranscriptional effect of IFN- on
COX-2 expression.
Interferon- is known to modulate the expression of a variety
of genes (reviewed in Ref. 38; note that the list of genes regulated by
IFN- are available at http://www.annurev.org./sup/material/htm).
Although this cytokine acts mostly as a positive regulator, several
constitutive and inducible genes such as IL-8 (39),
insulin-like growth factor (40), platelet endothelial cell
adhesion molecule (PECAM) (41), and low density
lipoprotein (LDL)-receptor related protein (42) are
susceptible to the inhibitory effect of IFN-. The transcriptional
influence of IFN- on susceptible genes is mediated through some
members of the Jak-STAT signaling pathway (43) and the IFN
regulatory factor (IRF) family (44). By nuclear run-on
analysis, we clearly demonstrated that IFN--primed macrophages
exhibit a lower rate of COX-2 gene transcription when the
cells were stimulated with IL-1ß, but not with LPS. A maximal
promoter activity has been reported from -1838 to +99 bp in the
5'-flanking region of the human COX-2 gene
(45). Although a variety of transcription factor elements
have been identified (45, 46), no IFN- activation site
(GAS) or IFN-stimulated response element sites have been reported. We
did a computer analysis on the sequence reported by Kosaka et al.
(46) of the 1.7-kb 5'-flanking region of the COX-2 gene
that revealed a putative GAS sequence that expands from -1391 to
-1383. An identical sequence has been described in the promoter region
of the murine Ly-6A/E gene (-1219 to -1211) that can bind STAT1
(47). GAS sequences are mainly related to positive
regulation of transcription (48). However, it has been
shown that GAS elements in the promoter region of the human IgE Ig can
also mediate repressor activity (49). Inhibition of
protein synthesis with CHX indicated that newly synthesized proteins
are required for the down-regulatory effect of IFN- on COX-2
expression. Although STAT proteins are constitutively expressed, it has
been shown that STAT1 expression can be up-regulated by IFN- in
human macrophages (50). It remains to be determined
whether this transcription factor binds to the distal GAS sequence on
the COX-2 promoter region or to other sequences and factors to mediate
IFN- repression.
In summary, we have presented evidence for the differential expression
of COX-2 mRNA, protein, and PGE2 production in naive and
IFN--primed macrophages stimulated with IL-1ß. Moreover, we showed
that IFN- down-regulated COX-2 expression at the transcriptional
level and had no effect on the stability of the transcripts. A clear
understanding of the molecular mechanisms that regulate COX-2
expression may lead to the development of novel treatments for
inflammation, colon cancer, and autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kris Chadee, Institute of Parasitology, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, P.Q., Canada, H9X 3V9. E-mail address:
3 Abbreviations used in this paper: COX, cyclooxygenase; AD, actinomycin D; CHX, cycloheximide; DEX, dexamethasone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EIA, enzyme immunoassay.
Received for publication February 5, 1998. Accepted for publication April 23, 1998.
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